Apoptosis assay was performed with apoptosis detection kit (BD, USA) as protocol. Cells (2 × 105 cells per well) were incubated in a six‐well plate and received different treatments for 48 hours; then cells were harvested and washed twice with ice‐cold PBS. apoptosis detection kit was used to measure the levels of apoptosis according to manufacturer's instructions by FCM (BD, USA). Data were analysed by FlowJo.
Apoptosis detection kit
Manufactured by BD
Sourced in United States, China, Germany
The Apoptosis detection kit is a laboratory product designed to identify and quantify apoptosis, a form of programmed cell death, in cell samples. The kit provides essential reagents and protocols to perform various assays for the detection and analysis of apoptotic cells.
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367 protocols using apoptosis detection kit
1
Apoptosis Quantification by Flow Cytometry
2
Anlotinib Inhibits Lung Cancer Cell
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Anlotinib (effective dose 12 mg/tablet, Chia Tai Tianqing Pharmaceutical Group Co., Ltd); DMEM high glucose medium (Gibco, USA); fetal bovine serum (HyClone, USA); apoptosis detection kit (Becton, Dickinson and Company, USA); cell cycle analysis kit (Beyotime Biotechnology Co., Ltd. China); human lung cancer H520, H226 and H2170 cell lines (provided by the Central Laboratory of Shandong Cancer Hospital); and linear accelerator (Elekta AB, Sweden).
3
Hyperthermia-Induced Apoptosis and Cell Cycle Analysis
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SW480, SW480-8HhP, SW480-CON, MKN74, MKN74-8HhP and MKN74-CON cells were seeded into 6-well plates and the hyperthermia group was treated at 43°C for 1 h every 48 h. For apoptotic analysis, apoptosis detection kit (Becton-Dickinson, Franklin Lakes, NJ, USA) was used to analyze apoptosis rates according to the manufacturer's instructions. For cell cycle analysis, cells growing in logarithmic phase were fixed with 75% ethanol for 24 h at −20°C, incubated with RNase A and Triton X-100 at 37°C for 30 min, and then incubated with propidium iodide at room temperature for 30 min. Cells were examined by flow cytometry, and CellQuest software (Becton-Dickinson) was used to conduct data acquisition and analysis.
4
Apoptosis Detection via Flow Cytometry
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Cells were stained with Apoptosis Detection Kit (Becton Dickinson, Franklin Lakes, USA) according to the manufacturer’s instructions. Cells were then tested on a FACScan flow cytometer (Becton Dickinson, Franklin Lakes, USA). All tests were performed in triplicate.
5
Cell Cycle and Apoptosis Analysis
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To determine the cell cycle distribution of cells, the cells were cultured in 12-well plates at a density of 1×105 cells/well. After 24 h, cells were transfected as described above. Twenty-four hours after transfection, cells were harvested by trypsinization, washed with PBS and fixed in 75% ice-cold ethanol overnight at 4°C. Afterwards, the cells were washed with PBS and incubated with 300 μL of staining solution (20 mg/mL propidium iodide and 10 U/mL RNase A) for 30 mins at room temperature. Cell cycle distributions were assessed by a fluorescence-activated cell sorting-based flow cytometer (Becton-Dickinson, USA).
Cell apoptosis analysis was performed with Annexin-V FITC Apoptosis Detection Kit (Life Technologies, USA), according to the manufacturer’s instructions. The cells were cultured in 12-well plates at a density of 1×105 cells/well, and transfected as described above for 48 h. After that, the cells were collected and stained with the Apoptosis Detection Kit and then examined by the flow cytometer (Becton-Dickinson, USA). The right lower quadrant indicates early apoptotic cells (Annexin V-FITC+/PI−) and the right upper quadrant (Annexin V-FITC+/PI+) indicates late apoptotic cells.
Cell apoptosis analysis was performed with Annexin-V FITC Apoptosis Detection Kit (Life Technologies, USA), according to the manufacturer’s instructions. The cells were cultured in 12-well plates at a density of 1×105 cells/well, and transfected as described above for 48 h. After that, the cells were collected and stained with the Apoptosis Detection Kit and then examined by the flow cytometer (Becton-Dickinson, USA). The right lower quadrant indicates early apoptotic cells (Annexin V-FITC+/PI−) and the right upper quadrant (Annexin V-FITC+/PI+) indicates late apoptotic cells.
6
Apoptosis Detection in Cell Lines
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DMEM medium and fetal calf serum (FCS) were purchased from GIBCO compnay (USA), The apoptosis detection kit was from Becton Dickinson and Company (San Jose, CA, USA). 3-(4,5-dimethyl-2-thiazyl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT), tyripsin and DMSO were from Amresco (USA), fix and perm kit was from Caltag laboratories, Beckman. Coulter USA), PE Anti-Bcl-2 Antibody was from Invitrogen Co., Ltd. (Shanghai, China), FITC Anti- Polyclonal antibodies for polymerase was from Bioss Technology Development Co., Ltd., (Beijing China). Other chemicals were commercially available reagent grade or ultrapure grade.
7
Cell Viability and Apoptosis Assay
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Viability was assessed by (3-(4,5-dimethylthiazol -2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H- tetrazolium) MTS assay, while the apoptotic cell rate by incubating treated cells with FITC–annexin V and Propidium iodide (Apoptosis detection kit, Becton Dickinson) followed by analysis on FACScantoII (Becton Dickinson).
8
Apoptosis Quantification by Flow Cytometry
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Cells were harvested, and apoptosis quantification was undertaken by flow cytometry using a Fluorescent Activated Cell Sorting (FACScan) Flow Cytometer (Becton Dickinson Immunocytometry Systems, San Jose, CA) equipped with an argon-ion laser (488 nm). Binding to annexin V and propidium iodide (PI) was determined using an Apoptosis Detection kit according to the manufacturer’s instructions (Becton Dickinson Pharmingen, Oxford, UK). To distinguish between apoptosis and necrosis, cells were double-stained with annexin V (green fluorescence) and PI (red fluorescence). Briefly, cells (100,000 cells/sample) were washed twice in cold PBS and suspended in binding buffer containing fluorescein isothiocyanate-conjugated annexin V (10 μg/ml) and PI (10 μg/ml). The cell suspension was incubated in the dark for 15 min, and signals were acquired by the flow cytometer. A total of 10,000 events were analyzed for each sample with Cell Quest software (Becton Dickinson Biosciences, San Jose, CA).
9
Quantifying Macrophage Apoptosis and Phenotypes
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RAW264.7 cells group as mentioned earlier. The apoptosis detection kit (556547, Becton Dickinson Company, United States) was used following manufacturer’s instructions. Shortly, cells at suitable concentrations were incubated with 5 μl FITC Annexin V and 5 μl PI for 15 min in dark. Then, we analyzed apoptosis by FACS within 1 h. Cells that were stained with FITC Annexin V (+) and PI(+) or (-) were considered apoptotic.
Cells were washed using 3 ml 1% BSA-PBS. Then, we suspended them in 1% BSA-PBS and counted their number with a cell counter, followed by pre-incubation with mouse TruStain FcX PLUS (anti-mouse CD16/32)(Biolegend, 156604, CA, USA)for 10 min. The cell suspension was stained with anti-mouse F4/80 BV605 (743281, Becton Dickinson, San Diego, CA, United States), APC anti-mouse CD163 (155,306, Biolegend, CA, United States), and anti-mouse CD86 BV421(105031, Biolegend, CA, United States) at 4°C for 30 min in the dark. After washing and resuspending in PBS, the cells were analyzed by FACS. The gating strategy was listed in (Supplementary Figure S1 ).
Cells were washed using 3 ml 1% BSA-PBS. Then, we suspended them in 1% BSA-PBS and counted their number with a cell counter, followed by pre-incubation with mouse TruStain FcX PLUS (anti-mouse CD16/32)(Biolegend, 156604, CA, USA)for 10 min. The cell suspension was stained with anti-mouse F4/80 BV605 (743281, Becton Dickinson, San Diego, CA, United States), APC anti-mouse CD163 (155,306, Biolegend, CA, United States), and anti-mouse CD86 BV421(105031, Biolegend, CA, United States) at 4°C for 30 min in the dark. After washing and resuspending in PBS, the cells were analyzed by FACS. The gating strategy was listed in (
10
Quantifying Apoptosis in TNBC Cells
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Apoptotic cell death in TNBC cells was quantified by flow cytometry using an apoptosis detection kit (Becton Dickinson, USA). The assay was performed as we previously described (Lou et al., 2009 (link)).
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